Parabacteroides distasonis regulates the infectivity and pathogenicity of SVCV at different water temperatures.

BACKGROUND: Spring viremia of carp virus (SVCV) infects a wide range of fish species and causes high mortality rates in aquaculture. This viral infection is characterized by seasonal outbreaks that are temperature-dependent. However, the specific mechanism behind temperature-dependent SVCV infectivity and pathogenicity remains unclear. Given the high sensitivity of the composition of intestinal microbiota to temperature changes, it would be interesting to investigate if the intestinal microbiota of fish could play a role in modulating the infectivity of SVCV at different temperatures. RESULTS: Our study found that significantly higher infectivity and pathogenicity of SVCV infection in zebrafish occurred at relatively lower temperature. Comparative analysis of the intestinal microbiota in zebrafish exposed to high- and low-temperature conditions revealed that temperature influenced the abundance and diversity of the intestinal microbiota in zebrafish. A significantly higher abundance of Parabacteroides distasonis and its metabolite secondary bile acid (deoxycholic acid, DCA) was detected in the intestine of zebrafish exposed to high temperature. Both colonization of Parabacteroides distasonis and feeding of DCA to zebrafish at low temperature significantly reduced the mortality caused by SVCV. An in vitro assay demonstrated that DCA could inhibit the assembly and release of SVCV. Notably, DCA also showed an inhibitory effect on the infectious hematopoietic necrosis virus, another Rhabdoviridae member known to be more infectious at low temperature. CONCLUSIONS: This study provides evidence that temperature can be an important factor to influence the composition of intestinal microbiota in zebrafish, consequently impacting the infectivity and pathogenicity of SVCV. The findings highlight the enrichment of Parabacteroides distasonis and its derivative, DCA, in the intestines of zebrafish raised at high temperature, and they possess an important role in preventing the infection of SVCV and other Rhabdoviridae members in host fish. Video Abstract.

The laminin receptor is a receptor for Micropterus salmoides rhabdovirus.

Micropterus salmoides rhabdovirus (MSRV) is an important pathogen of largemouth bass. Despite extensive research, the functional receptors of MSRV remained unknown. This study identified the host protein, laminin receptor (LamR), as a cellular receptor facilitating MSRV entry into host cells. Our results demonstrated that LamR directly interacts with MSRV G protein, playing a pivotal role in the attachment and internalization processes of MSRV. Knockdown of LamR with siRNA, blocking cells with LamR antibody, or incubating MSRV virions with soluble LamR protein significantly reduced MSRV entry. Notably, we found that LamR mediated MSRV entry via clathrin-mediated endocytosis. Additionally, our findings revealed that MSRV G and LamR were internalized into cells and co-localized in the early and late endosomes. These findings highlight the significance of LamR as a cellular receptor facilitating MSRV binding and entry into target cells through interaction with the MSRV G protein. IMPORTANCE: Despite the serious epidemic caused by Micropterus salmoides rhabdovirus (MSRV) in largemouth bass, the precise mechanism by which it invades host cells remains unclear. Here, we determined that laminin receptor (LamR) is a novel target of MSRV, that interacts with its G protein and is involved in viral attachment and internalization, transporting with MSRV together in early and late endosomes. This is the first report demonstrating that LamR is a cellular receptor in the MSRV life cycle, thus contributing new insights into host-pathogen interactions.

Selective autophagy receptor p62 promotes antibacterial and antiviral immunity in common carp (Cyprinus carpio).

Sequestosome 1 (SQSTM1/p62) is a selective autophagy adapter protein that participates in antiviral and bacterial immune responses and plays an important regulatory role in clearing the proteins to be degraded and maintaining intracellular protein homeostasis. In this study, two p62 genes were cloned from common carp (Cyprinus carpio), namely Ccp62-1 and Ccp62-2, and conducted bioinformatics analysis on them. The results showed that Ccp62s had the same structural domain (Phox and Bem1 domain, ZZ-type zinc finger domain, and ubiquitin-associated domain) as p62 from other species. Ccp62s were widely expressed in various tissues of fish, and highly expressed in immune organs such as gills, spleen, head kidney, etc. Subcellular localization study showed that they were mainly distributed in punctate aggregates in the cytoplasm. After stimulation with Aeromonas hydrophila and spring viraemia of carp virus (SVCV), the expression level of Ccp62s was generally up-regulated. Overexpression of Ccp62s in EPC cells could inhibit SVCV replication. Upon A. hydrophila challenge, the bacterial load in Ccp62s-overexpressing group was significantly reduced, the expression levels of pro-inflammatory cytokines and interferon factors were increased, and the survival rate of the fish was improved. These results indicated that Ccp62s were involved in the immune response of common carp to bacterial and viral infections.

RING finger protein 122-like (RNF122L) negatively regulates antiviral immune response by targeting STING in common carp (Cyprinus carpio L.).

Stimulator of interferon genes (STING), as an imperative adaptor protein in innate immune, responds to nucleic acid from invading pathogens to build antiviral responses in host cells. Aberrant activation of STING may trigger tissue damage and autoimmune diseases. Given the decisive role in initiating innate immune response, the activity of STING is intricately governed by several posttranslational modifications, including phosphorylation and ubiquitination. Here, we cloned and characterized a novel RNF122 homolog from common carp (named CcRNF122L). Expression analysis disclosed that the expression of CcRNF122L is up-regulated under spring viremia of carp virus (SVCV) stimulation in vivo and in vitro. Overexpression of CcRNF122L hampers SVCV- or poly(I:C)-mediated the expression of IFN-1 and ISGs in a dose-dependent way. Mechanistically, CcRNF122L interacts with STING and promotes the polyubiquitylation of STING. This polyubiquitylation event inhibits the aggregation of STING and the subsequent recruitment of TBK1 and IRF3 to the signaling complex. Additionally, the deletion of the TM domain abolishes the negative regulatory function of CcRNF122L. Collectively, our discoveries unveil a mechanism that governs the STING function and the precise adjustment of the innate immune response in teleost.

Tissue factor pathway inhibitors disrupt structures of rhabdovirus/ranairidovirus and inhibit viral infection in Chinese perch, Siniperca chuatsi.

Viral diseases have caused great economic losses to the aquaculture industry. However, there are currently no specific drugs to treat these diseases. Herein, we utilized Siniperca chuatsi as an experimental model, and successfully extracted two tissue factor pathway inhibitors (TFPIs) that were highly distributed in different tissues. We then designed four novel peptides based on the TFPIs, named TS20, TS25, TS16, and TS30. Among them, TS25 and TS30 showed good biosafety and high antiviral activity. Further studies showed that TS25 and TS30 exerted their antiviral functions by preventing viruses from invading Chinese perch brain (CPB) cells and disrupting Siniperca chuatsi rhabdovirus (SCRV)/Siniperca chuatsi ranairidovirus (SCRIV) viral structures. Additionally, compared with the control group, TS25 and TS30 could significantly reduce the mortality of Siniperca chuatsi, the relative protection rates of TS25 against SCRV and SCRIV were 71.25 % and 53.85 % respectively, and the relative protection rate of TS30 against SCRIV was 69.23 %, indicating that they also had significant antiviral activity in vivo. This study provided an approach for designing peptides with biosafety and antiviral activity based on host proteins, which had potential applications in the prevention and treatment of viral diseases.

Fish ubiquitin-specific protease 8 (USP8) inhibits IFN production through autophagy-lysosomal dependent degradation of IRF7.

Interferon regulatory factor 7 (IRF7) is considered the master regulator of virus-induced interferon (IFN) production. However, to avoid an autoimmune response, the expression of IRF7 must be tightly controlled. In this study, we report that zebrafish ubiquitin-specific protease 8 (USP8) promotes IRF7 degradation through an autophagy-lysosome-dependent pathway to inhibit IFN production. First, zebrafish usp8 is induced upon spring viremia of carp virus (SVCV) infection and polyinosinic/polycytidylic acid (poly I:C) stimulation. Second, overexpression of USP8 suppresses SVCV or poly I:C-mediated IFN expression. Mechanistically, USP8 interacts with IRF7 and promotes its degradation via an autophagy-lysosome-dependent pathway. Finally, USP8 significantly suppresses cellular antiviral responses and enhances SVCV proliferation. In summary, our discoveries offer a perspective on the role of zebrafish USP8 and provide additional understanding of the regulation of IRF7 in host antiviral immune response.

USP14 negatively regulates IFN signaling by dampening K63-linked ubiquitination of TBK1 in black carp.

USP14 regulates the immune related pathways by deubiquitinating the signaling molecules in mammals. In teleost, USP14 is also reported to inhibit the antiviral immune response through TBK1, but its regulatory mechanism remains obscure. To elucidate the role of USP14 in the RLR/IFN antiviral pathway in teleost, the homolog USP14 (bcUSP14) of black carp (Mylopharyngodon piceus) has been cloned and characterize in this paper. bcUSP14 contains 490 amino acids (aa), and the sequence is well conserved among in vertebrates. Over-expression of bcUSP14 in EPC cells attenuated SVCV-induced transcription activity of IFN promoters and enhanced SVCV replication. Knockdown of bcUSP14 in MPK cells led to the increased transcription of IFNs and decreased SVCV replication, suggesting the improved antiviral activity of the host cells. The interaction between bcUSP14 and bcTBK1 was identified by both co-immunoprecipitation and immunofluorescent staining. Co-expressed bcUSP14 obviously inhibited bcTBK1-induced IFN production and antiviral activity in EPC cells. K63-linked polyubiquitination of bcTBK1 was dampened by co-expressed bcUSP14, and bcTBK1-mediated phosphorylation and nuclear translocation of IRF3 were also inhibited by this deubiquitinase. Thus, all the data demonstrated that USP14 interacts with and inhibits TBK1 through deubiquitinating TBK1 in black carp.

Autophagy mediated degradation of MITA/TBK1/IRF3 by a hnRNP family member attenuates interferon production in fish.

HnRNP A/B belongs to the heterogeneous nuclear ribonucleoprotein (hnRNP) family and plays an important role in regulating viral protein translation and genome replication. Here, we found that overexpression of hnRNP A/B promoted spring viremia of carp virus (SVCV) and cyprinid herpesvirus 3 (CyHV3) replication. Further, hnRNP A/B was shown to act as a negative regulator of type I interferon (IFN) response. Mechanistically, hnRNP A/B interacted with MITA, TBK1 and IRF3 to initiate their degradation. In addition, hnRNP A/B bound to the kinase domain of TBK1, the C terminal domain of MITA and IAD domain of IRF3, and the RRM1 domain of hnRNP A/B bound to TBK1, RRM2 domain bound to IRF3 and MITA. Our study provides novel insights into the functions of hnRNP A/B in regulating host antiviral response.

Innate immune response of snakehead fish to Indian strain of snakehead rhabdovirus (SHRV-In) infection and the infectivity potential of the virus to other freshwater fishes.

A new virus known as snakehead rhabdovirus (SHRV-In) was discovered in South India in striped snakehead (Channa striata) that had hemorrhagic patches and cutaneous ulcerations. The virus is the most potentially harmful pathogen of snakehead because it could cause 100% mortality within 5 days. The goal of the current investigation was to evaluate the infectivity of rhabdovirus in freshwater fishes and to analyze the immune response in snakehead fish after challenge with SHRV-In. The infectivity study of SHRV-In against three freshwater fish such as tilapia, grass carp and loach showed that the virus could not induce mortality in any of them. Snakehead fish challenged with SHRV-In showed significant (p < 0.05) changes in haematological parameters such as red blood cell (RBC), haemoglobin (HGB), haematocrit (HCT), mean corpuscular haemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), white blood cell (WBC), total platelet (PLT) counts, mean platelet volume (MPV) and immunological markers such as respiratory burst, superoxide dismutase, catalase activity and myeloperoxidase activity at 6, 12, 24 and 48 hpi. Real time PCR was executed to examine the expression profile of innate immune genes such as IRF-7, IL-8 and IL-12 in Snakehead fish at 6, 12, 24 and 48 h post SHRV-In infection. Immune gene expression of IRF-7, IL-8 and IL-12 were up-regulated in the spleen when compared to kidney at 6 and 12 hpi. However, the expression level of all the genes was down-regulated at 24 and 48 hpi. The down regulation of innate immune genes after 24 hpi in these tissues may be the result of increased multiplication of SHRV-In by interfering with the immune signaling pathway.

Establishment and characterization of a kidney cell line from hybrid snakehead (male Channa argus × female Channa maculata) and its susceptibility to hybrid snakehead rhabdovirus (HSHRV).

Hybrid snakehead (male Channa argus × female Channa maculata) is an emerging fish breed with increasing production levels. However, infection with hybrid snakehead rhabdovirus (HSHRV) critically affects hybrid snakehead farming. In this study, a fish cell line called CAMK, derived from the kidneys of hybrid snakehead, was established and characterized. CAMK cells exhibited the maximum growth rate at 28 °C in Leibovitz's-15 medium supplemented with 10% fetal bovine serum(FBS). Karyotyping revealed diploid chromosomes in 54% of the cells at the 50th passage (2n = 66), and 16S rRNA sequencing validated that CAMK cells originated fromhybrid snakehead, and the detection of kidney-specific antibodies suggested that it originated from kidney. .The culture was free from mycoplasma contamination, and the green fluorescent protein gene was effectively transfected into CAMK cells, indicating their potential use for in vitro gene expression investigations. Furthermore, qRT-PCR and immunofluorescence analysis revealed that HSHRV could replicate in CAMK cells, indicating that the cells were susceptible to the virus. Transmission electron microscopy revealed that the viral particles had bullet-like morphology. The replication efficiency of HSHRV was 107.33 TCID50/mL. Altogether, we successfully established and characterized a kidney cell line susceptible to the virus. These findings provide a valuable reference for further genetic and virological studies.

RNAi-mediated knockdown of exportin 1 negatively affected ovary development, survival and maize mosaic virus accumulation in its insect vector Peregrinus maidis.

Exportin 1 (XPO1) is the major karyopherin-ß nuclear receptor mediating the nuclear export of hundreds of proteins and some classes of RNA and regulates several critical processes in the cell, including cell-cycle progression, transcription and translation. Viruses have co-opted XPO1 to promote nucleocytoplasmic transport of viral proteins and RNA. Maize mosaic virus (MMV) is a plant-infecting rhabdovirus transmitted in a circulative propagative manner by the corn planthopper, Peregrinus maidis. MMV replicates in the nucleus of plant and insect hosts, and it remains unknown whether MMV co-opts P. maidis XPO1 (PmXPO1) to complete its life cycle. Because XPO1 plays multiple regulatory roles in cell functions and virus infection, we hypothesized that RNAi-mediated silencing of XPO1 would negatively affect MMV accumulation and insect physiology. Although PmXPO1 expression was not modulated during MMV infection, PmXPO1 knockdown negatively affected MMV accumulation in P. maidis at 12 and 15 days after microinjection. Likewise, PmXPO1 knockdown negatively affected P. maidis survival and reproduction. PmXPO1 exhibited tissue-specific expression patterns with higher expression in the ovaries compared with the guts of adult females. Survival rate was significantly lower for PmXPO1 knockdown females, compared with controls, but no effect was observed for males. PmXPO1 knockdown experiments revealed a role for PmXPO1 in ovary function and egg production. Oviposition and egg hatch on plants were dramatically reduced in females treated with dsRNA PmXPO1. These results suggest that PmXPO1 is a positive regulator of P. maidis reproduction and that it plays a proviral role in the insect vector supporting MMV infection.

Black carp ATG16L1 negatively regulates STING-mediated antiviral innate immune response.

The precise control of interferon (IFN) production is indispensable for the host to eliminate invading viruses and maintain a homeostatic state. In mammals, stimulator of interferon genes (STING) is a prominent adaptor involved in antiviral immune signaling pathways. However, the regulatory mechanism of piscine STING has not been thoroughly investigated. Here, we report that autophagy related 16 like 1 (bcATG16L1) of black carp (Mylopharyngodon piceus) is a negative regulator in black carp STING (bcSTING)-mediated signaling pathway. Initially, we substantiated that knockdown of bcATG16L1 increased the transcription of IFN and ISGs and enhanced the antiviral activity of the host cells. Subsequently, we identified that bcATG16L1 inhibited the bcSTING-mediated IFN promoter activation and proved that bcATG16L1 suppressed bcSTING-mediated antiviral ability. Furthermore, we revealed that bcATG16L1 interacted with bcSTING and the two proteins shared a similar subcellular distribution. Mechanically, we found that bcATG16L1 attenuated the oligomerization of bcSTING, which was a key step for bcSTING activation. Taken together, our results indicate that bcATG16L1 interacts with bcSTING, dampens the oligomerization of bcSTING, and negatively regulates bcSTING-mediated antiviral activity.

GSTP1 is a negative regulator of MAVS in the antiviral signaling against SVCV infection.

Glutathione S-transferase P1 (GSTP1), the most ubiquitous member of the GST superfamily, plays vital roles in the detoxification, antioxidant defense, and modulation of inflammatory responses. However, limited studies have been conducted on the function of GSTP1 in antiviral innate immunity. In this study, we have cloned the homolog of GSTP1 in triploid hybrid crucian carp (3nGSTP1) and investigated its regulatory role in the interferon signaling pathway. The open reading frame of 3nGSTP1 is composed of 627 nucleotides, encoding 209 amino acids. In response to spring viremia of carp virus (SVCV) infection, the mRNA level of 3nGSTP1 was up-regulated in the liver, kidney, and caudal fin cell lines (3 nF C) of triploid fish. The knockdown of 3nGSTP1 in 3 nF C improved host cell's antiviral capacity and attenuated SVCV replication. Additionally, overexpression of 3nGSTP1 inhibited the activation of IFN promoters induced by SVCV infection, poly (I:C) stimulation, or the RLR signaling factors. The co-immunoprecipitation assays further revealed that 3nGSTP1 interacts with 3nMAVS. In addition, 3nGSTP1 dose-dependently inhibited 3nMAVS-mediated antiviral activity and reduced 3nMAVS protein level. Mechanistically, 3nGSTP1 promoted ubiquitin-proteasome degradation of MAVS by promoting its K48-linked polyubiquitination. To conclude, our results indicate that GSTP1 acts as a novel inhibitor of MAVS, which negatively regulates the IFN signaling.

Effects on viral suppression and the early-immune expression of ribavirin against spring viremia of carp virus in vitro.

Spring viremia of carp virus (SVCV) is a globally distributed virus that causes severe clinical symptoms and high mortality in fish belonging to the families Cyprinidae and Siluridae. To protect the host against viral infection, understanding the relatedness between viral susceptibility and antiviral mechanisms must be crucial. Thus, we evaluated the viral suppression efficacy of ribavirin by measuring the transcription levels of viral and immune genes in vitro. The results showed that following ribavirin treatment after SVCV infection (MOI 0.1), ribavirin inhibited SVCV replication in epithelioma papulosum cyprini (EPC) cells and completely inhibited viral gene (G and N) expression at concentrations above 10 µg/mL at 48 h post-infection. Ribavirin does not directly damage SVCV particles but inhibits early viral replication. In the absence of SVCV infection, the immunological dynamics triggered by ribavirin resulted in upregulated pattern recognition receptors and proinflammatory cytokine-related genes (i.e., PI3K, MYD88, IRAK1, RIG-І, MAVS, Mx1, TNF-α, and NF-κB). Furthermore, EPC cells treated with ribavirin following SVCV infection showed upregulation of PI3K, MYD88, IRAK1, RIG-І, TNF-α, and NF-κB genes within 24 h post-SVCV infection, suggesting that ribavirin positively inhibits the SVCV infection in vitro.

Characterization of STING from common carp (Cyprinus carpio L.) involved in spring viremia of carp virus infection.

Stimulator of interferon genes (STING) serve as an endoplasmic reticulum (ER) protein and modulates innate immune responses to viral contagion. Most investigations involving teleost STING antiviral immunity have examined DNA viruses. Therefore, fish STING signaling events against RNA viruses require additional exploration. Here, common carp STING (named CcSTING) was cloned and characterized. The bioinformatics analyses of CcSTING showed evolutionary conservations and were most closely related to other cyprinid STINGs. Immunofluorescence staining discovered that the CcSTING was chiefly placed in the cytoplasm, specifically within the ER. CcSTING was ubiquitously generated in all analyzed organs, with especially strong expression in the gills and head kidney. Spring viremia of carp virus (SVCV) stimulation and poly(I:C) infection induced the generation of CcSTING in immune-associated organs, as well as in peripheral blood leukocytes. Additional investigations revealed that CcSTING overexpression strongly suppressed SVCV replication in EPC cells. Mechanistically, CcSTING enhanced IFN-1 and ISGs expression following SVCV infection. CcSTING also substantially increased both IFN and NF-κB promoter luciferase activity via a dosage-dependent fashion. Lastly, CcSTING significantly up-regulated both TBK1 and p65 phosphorylation. Collectively, these findings demonstrated the critical role and underlying mechanism of fish STING in response to RNA virus.

Eukaryotic translation elongation factor 1 alpha (eEF1A) inhibits Siniperca chuatsi rhabdovirus (SCRV) infection through two distinct mechanisms.

IMPORTANCE: Although a virus can regulate many cellular responses to facilitate its replication by interacting with host proteins, the host can also restrict virus infection through these interactions. In the present study, we showed that the host eukaryotic translation elongation factor 1 alpha (eEF1A), an essential protein in the translation machinery, interacted with two proteins of a fish rhabdovirus, Siniperca chuatsi rhabdovirus (SCRV), and inhibited virus infection via two different mechanisms: (i) inhibiting the formation of crucial viral protein complexes required for virus transcription and replication and (ii) promoting the ubiquitin-proteasome degradation of viral protein. We also revealed the functional regions of eEF1A that are involved in the two processes. Such a host protein inhibiting a rhabdovirus infection in two ways is rarely reported. These findings provided new information for the interactions between host and fish rhabdovirus.

Fish Rhbdd3 positively regulates IFN response through RIG-I signaling pathway.

Rhomboid domain-containing protein 3 (Rhbdd3) is a member of the rhomboid family, which can modulate the innate immune response in mammals. Nonetheless, the function and regulatory mechanism of fish Rhbdd3 during viral infection have not been characterized. In this study, Rhbdd3 was firstly cloned from common carp (Cyprinus carpio) and nominated as CcRhbdd3. Phylogenetically characterization showed that CcRhbdd3 shared a relatively long evolutionary distance with its mammalian homologs. In vivo experiment demonstrated that spring viraemia of carp virus (SVCV) infection promoted the expression of CcRhbdd3 in the liver, spleen, kidney and muscle tissues. Furthermore, overexpression of CcRhbdd3 significantly inhibited SVCV propagation, whereas knockdown of CcRhbdd3 markedly promoted SVCV replication in susceptible cells. RNA-seq and following validation showed that CcRhbdd3 overexpression upregulated the expression of several RIG-I signaling related genes, including TRIM25, TRAF2, MDA5, LGP2, IFN1, IFN3, RIG-I, IRF3 and ISG15. Moreover, CcRhbdd3 promoted the expression of NF-κB, a central immune regulator. Subcellular localization experiments showed that CcRhbdd3 was primarily distributed in the cytoplasm and co-localized with Rab5 in the early endosomes. Truncation experiments further demonstrated that the C-terminus containing the ubiquitin-binding associated domain, was crucial for both the subcellular localization and antiviral activity of CcRhbdd3. The findings in this study provide new insight into the host antiviral mechanism against aquatic RNA virus infection, and will facilitate the development of therapeutic strategies for the infection of SVCV.

Application of rhein as an immunostimulant controls spring viremia of carp virus infection.

In recent years, the exploration of natural compounds possessing both immunostimulatory and antiviral activities has attracted growing attention in aquaculture research. Consequently, the pursuit of identifying natural products exhibiting anti-SVCV potential as immunostimulants holds significant promise, offering a pathway to mitigate the economic ramifications inflicted by SVCV outbreaks in aquaculture settings. Among them, rhein emerges as a particularly compelling contender. Boasting a widespread distribution, well-established extraction methods, and multiple biological activities, it has exhibited the capacity to enhance the antiviral activity of host cells in vitro by blocking the viral internalization process, with a peak inhibition rate of 44.0%. Based on this intervention, rhein inhibited apoptosis and mitochondrial damage triggered by SVCV infection, ultimately producing a significant antiviral effect. Moving beyond the laboratory setting, rhein's efficacy translates effectively into in vivo scenarios. It has demonstrated substantial antiviral potency by increasing the expression of antiviral-related genes, most notably, retinoic acid-inducible gene I (RIG-I), interferon-φ (IFN-φ) and IFN-stimulated gene product 15 (ISG15). In concert with this genetic modulation, rhein efficiently reduces the viral load, precipitating a consequential enhancement in the survival rate of SVCV-infected fish, elevating it to an encouraging 16%. In conclusion, the outcomes of our investigation offer a compelling testament to rhein's potential as a valuable immunomodulator in the battle against SVCV infections in aquaculture, and the remarkable attributes exhibited by rhein underscore its viability for future commercial deployment.

Black carp RIOK3 suppresses MDA5-mediated IFN signaling in the antiviral innate immunity.

In mammals, right open reading frame kinase 3 (RIOK3) is related with cancer development and immune regulation. To explore the role of teleost RIOK3 in the antiviral innate immunity, the homolog of RIOK3 (bcRIOK3) from black carp (Mylopharyngodon piceus) has been cloned and characterized in this study. Sequence analysis revealed that bcRIOK3 is conserved in vertebrates. The transcription of bcRIOK3 varied in host cells in response to the stimulation of spring viremia of carp virus (SVCV), poly (I:C), and LPS. Immunoblotting (IB) and immunofluorescence (IF) assays identified bcRIOK3 as a cytoplasmic protein with a molecular weight of ∼60 kDa. It was interesting that bcRIOK3 knockdown led to the decreased basal mRNA levels of IFNa, IFNb and Viperin; however, triggered obviously higher mRNA levels of the above genes after viral infection and enhanced host resistance to SVCV. Like its mammalian counterpart, bcRIOK3 overexpression in EPC cells showed a significant inhibitory effect on black carp MDA5 (bcMDA5)-mediated transcription of interferon promoters and antiviral activity. Co-immunoprecipitation and immunofluorescent assays identified the association between bcRIOK3 and bcMDA5. Further analysis revealed that bcRIOK3 enhanced the K48-linked ubiquitination and proteasome-dependent degradation of bcMDA5, and it weakened the oligomerization of bcMDA5 under poly (I:C) stimulation. In summary, our data conclude that RIOK3 dampens MDA5-mediated IFN signaling by promoting its degradation in black carp, which provide new insights into the regulation of IFN signaling in teleost.

hnRNPM suppressed IRF7-mediated IFN signaling in the antiviral innate immunity in triploid hybrid fish.

Mammalian heterogeneous nuclear ribonucleoproteins M (hnRNPM) is a critical splicing regulatory protein that has been reported to negatively regulate the RLR signaling pathway by impairing the binding of RIG-I and MDA5 to viral RNA. To explore the role of hnRNPM in the antiviral innate immune response in teleost fish, the hnRNPM homologue of triploid fish (3nhnRNPM) has been cloned and identified in this paper. The CDS of 3nhnRNPM gene is composed of 2016 nucleotides and encodes 671 amino acids. 3nhnRNPM migrated around 71 kDa in immunoblotting assay and was mainly detected in the nucleus in nucleo-cytoplasmic separation assay and immunofluorescent staining test. When 3nhnRNPM and 3nIRF7 were co-expressed in EPC cells, 3nhnRNPM significantly reduced the 3nIRF7-induced interferon (IFN) promoter transcription. Correspondingly, the mRNA levels of the SVCV-M, -N, -P, and -G genes were noteworthily enhanced, but the transcription levels of epcIFNφ1, epcMx1, epcPKR, and epcISG15 were dramatically decreased. Additionally, the knockdown of 3nhnRNPM resulted in restricted SVCV replication and enhanced host cell antiviral activity. Furthermore, the association between 3nhnRNPM and 3nIRF7 has been identified by the co-immunoprecipitation assay. In addition, we found that 3nIRF7 was detained in the nucleus when co-expressed with 3nhnRNPM. To sum up, our data supported the conclusion that 3nhnRNPM suppressed 3nIRF7-mediated IFN signaling in the antiviral innate immunity.